In vertebrates, a functional lens requires the ability of lens fiber cells to differentiate, elongate, and express abundant levels of the appropriate crystallin molecules. Factors which affect any of these processes are detrimental and can lead to the formation of cataract disease. Knockout studies have shown that the mouse transcription factor Proxl regulates lens fiber elongation and crystallin gene expression. Unfortunately, Proxl knockout animals die by embryonic day 13.5, making further analysis of this phenotype difficult. Our preliminary data demonstrate that the Drosophila ortholog of Proxl, called Prospero (Pros), is also necessary for lens formation. Similar to Proxl, Pros affects lens cell differentiation and crystallin gene expression. This is one of the first reports that lens development in invertebrates and vertebrates shares similar transcriptional regulatory pathways. Interestingly, Pros and Proxl also exhibit both nuclear and cytoplasmic expression during lens cell differentiation, suggesting a non-transcriptional role for these proteins as well. The studies outlined here are aimed to explore the evolutionary conserved basis for Pros/Prox1-mediated lens development. For this, we will: 1) more precisely characterize the role for Pros in lens cell development, 2) test the contribution of Pros intracellular localization in lens development, and 3) characterize functional conservation with the Pros/Prox1 family of transcription factors. These studies will use a combination of in vitro biochemical and transcriptional regulatory assays, site-directed mutagenesis, and in vivo genetic analyses to provide a broad perspective on how Pros/Prox1 proteins function during lens development. This work will provide the framework necessary to perform more in-depth genetic studies aimed at identifying key regulatory pathways necessary for normal lens development as a means for understanding what aspects of these pathways are disturbed in patients suffering from congenital and age- related cataracts. LAY: Cataracts result from the failure of our lens to properly develop or to be maintained. Unfortunately, not much is known about how a normal lens develops, making it difficult for us to devise ways to prevent abnormal lens formation such as in cataract disease. This proposal is focused on developing a new genetic model that will allow us to rapidly identify new pathways that are necessary for lens development as a means to circumvent events that lead to cataract formation and blindness.